Combined Inhibition of Bcl-2 and Mcl-1 Circumvents Resistance of TP53 Deficient/Mutant AML to BH3 Mimetics

Abstract AML patients with TP53 mutations have extremely poor clinical outcomes. This is due primarily to limited responses to available therapies including the highly promising FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents (DiNardo CD et al., Blood 2020), which resulted in CR/CRi rates of 70-95% and good tolerability in elderly patients (DiNardo CD et al., Lancet Oncol 2018 and Blood 2019). Apoptosis is regulated by anti- and pro-apoptotic proteins. While p53 does not directly regulate anti-apoptotic Bcl-2 proteins that are resistance factors for VEN, p53 transcriptionally up-regulates pro-apoptotic Bcl-2 proteins. Reverse phase protein array analysis of samples from newly-diagnosed AML patients found that pro-apoptotic Bax was significantly decreased in patients with TP53 mutations (Carter BZ, ASH 2019), which, as expected, diminished the effectiveness of Bcl-2 inhibition. Thus, strategies to target additional anti-apoptotic proteins, or increase pro-apoptotic proteins, are needed to enhance the efficacy of Bcl-2 inhibition in these patients. We determined protein levels of Bcl-2 family members in isogeneic Molm13 cells with TP53-knockout (KO), or with various hotspot TP53 mutations including R175H, Y220C, M237I, R248Q, R273H, and R282W. We observed markedly decreased Bax expression, to a less degree Bak decrease, and variable alterations in other Bcl-2 proteins in these cells compared to TP53-wild-type (WT) controls. We treated the aforementioned cells with VEN or the Mcl-1 inhibitor AMG 176 and found that TP53-KO or mutant cells were more resistant to both VEN and AMG 176 compared to WT controls. However, the combination of two inhibitors was highly synergistic in both settings, controls (CI = 0.2) and TP53-KO and mutant cells (CI < 0.1). To demonstrate that the decreased sensitivity to BH3 mimetics was, at least in part, mediated through Bax reduction in the TP53-mutant cells, we treated Bax knockdown (KD) Molm13 cells with VEN, AMG 176, or both. The Bax KD cells were resistant to VEN and AMG 176, while the combination of the two agents synergistically induced cell death. To establish potential clinical relevance of co-targeting Bcl-2 and Mcl-1 in TP53-mutant AML, we co-cultured cells from various TP53-mutant AML patients (n = 8) with mesenchymal stromal cells and treated them with VEN, AMG 176, or both. The combination synergistically induced cell death in both CD45 + leukemia blasts (CI values between 0.04 ± 0.04 to 0.34 ± 0.10) and CD34 + AML stem/progenitor cells (CI values between 0.07 ± 0.08 to 0.28 ± 0.14). RNA-sequencing of mononuclear and MRD cells of clinical samples (Issa G, ASH 2019) collected after induction therapy revealed that Mcl-1 expression was significantly higher in the TP53-mutated mononuclear and MRD cells compared to their WT counterparts (Fig. 1), which suggests that Mcl-1 contributes to treatment resistance and disease relapse. This further suggests that Mcl-1 inhibition should be incorporated in AML treatment, including VEN-based therapies, for patients with TP53 mutations. Finally, we treated NSG mice inoculated with isogeneic TP53-WT luciferase/GFP-labeled Molm13 and BFP-labeled TP53 R248W/R213* Molm13 cells (10:1) with VEN, AMG 176, or their combination. Only the combination treatment markedly decreased the number of GFP- and BFP-labeled cells in circulation and significantly prolonged mouse survival (median 23 d, 25 d, 24.5 d for control, VEN, AMG 176, respectively; and 45 d for VEN + AMG 176: P = 0.0007, 0.0009, and 0.0011 of combination vs. control, VEN, and AMG 176, respectively) (Fig. 2). Collectively, we demonstrate that decreased Bax contributes to resistance of TP53-mutant AML to BH3 mimetics. Mcl-1 expression positively impacts therapy resistance and disease reoccurrence in TP53-mutant AML. Thus, targeting Bcl-2 or Mcl-1 individually is insufficient and inhibition of both proteins is needed to shift cell fate from survival to death and circumvents resistance of TP53 deficient/mutant AML and AML stem/progenitor cells to BH3 mimetics. The concept warrants further clinical evaluation. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Andreeff: Medicxi: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Syndax: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; Glycomimetics: Consultancy; Oxford Biomedica UK: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

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TP53 Deficient/Mutant AMLs Are Resistant to Individual BH3 Mimetics: High Efficacy of Combined Inhibition of Bcl-2 and Mcl-1

Blood ◽

10.1182/blood-2019-124826 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1271-1271 ◽

Cited By ~ 1

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Steven M. Kornblau ◽

Wenjing Tao ◽

Yuki Nishida ◽

...

Keyword(s):

Protein Expression ◽

Board Of Directors ◽

Research Funding ◽

Deficient Mutant ◽

Wild Type ◽

Advisory Committees ◽

Bh3 Mimetics ◽

Tp53 Mutations ◽

Bax Protein ◽

Equity Ownership

In spite of recent progress in AML therapy, the outcomes of TP53 mutated AML remain extremely poor. The FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents is resulting in CR/CRi rates of 70-95% and good tolerability in elderly AML patients. However, patients with TP53 mutations achieve lower response rates (CR/CRi 47%) (DiNardo CD et al., Lancet Oncol 2018; DiNardo CD et al., Blood 2019) and invariably relapse. Combined Bcl-2 inhibition and p53 activation is synthetic lethal in TP53 wild-type AML in part through targeting Mcl-1 (Pan R et al., Cancer Cell 2016). In TP53 deficient/mutant AML, direct targeting of Mcl-1 may partially compensate for the TP53 defect. We therefore postulate that combined inhibition of Mcl-1 and Bcl-2 effectively induces apoptosis in TP53 deficient/mutant AML cells. Reverse phase protein array analysis of a large cohort of newly diagnosed AML patients (n=511) enabled us to stratify patients into various prognostic groups based on p53 pathway protein expression, which included 8 core proteins: TP53, TP53pS15, MDM2, MDM4, TRIM24, SFN, IRS1.pS1101, and YWHAZ. The group with p53 pathway dysfunction, defined by high p53 protein levels, is characterized by poor outcomes (Quintas-Cardama A et al., Leukemia 2017). This group, encompassing both TP53 wild-type and TP53 mutations, had significantly lower expression of Bax (p=0.0007), the chief executioner of intrinsic apoptosis. A survey of Bcl-2 family proteins in TP53 wild-type and mutant AML showed that only Bax was significantly lower in patients with TP53 mutations (p=0.0498). We next investigated the roles of p53 in response to BH3 mimetics in TP53 wild-type and knockdown (KD) OCI-AML3 cells and in TP53 wild-type and mutant Molm13 cells, generated by long-term exposure of TP53 wild-type Molm13 cells to RG7388 (idasanutlin). Western blot analysis showed that Bax protein was consistently decreased in both TP53 KD and mutant cell lines compared to their respective controls. p53 KD OCI-AML3 and TP53 mutant Molm13 cells exhibited decreased sensitivity not only to VEN, but also to Mcl-1 inhibitor AZD5991 compared to OCI-AML3 vector control and Molm13 parental cells, respectively. To investigate if combined inhibition of Bcl-2 and Mcl-1 could counter-balance the loss of TP53 activity, p53 KD and TP53 mutated AML cells were treated with VEN and AZD5991: like in their respective control cells, the combination synergistically induced cell death in these cells (Fig. 1). The EC50 levels of VEN required for the described synergism in OCI-AML3 cells can easily be reached/exceeded clinically. The combination was also synergistic in leukemia cell lines lacking wild-type TP53 such as KG-1 and U937. Importantly, the combined inhibition was more effective than each single agent in primary AML cells and stem/progenitor cells lacking wild-type TP53 due to deletion of chromosome 17 or mutations in TP53 gene. Conclusion: AML cells deficient in functional TP53 have decreased Bax protein expression, increased apoptotic threshold and are more resistant to individual BH3 mimetics. Combined inhibition of Bcl-2 and Mcl-1 is highly synergistic in p53 deficient/mutant AML. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy.

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Expression of PD-L1 in Leukemic Progenitor Cells Defines NPM1 Mutated AML As a Potential Subgroup for PD1/PD-L1 Directed Immunotherapy

Blood ◽

10.1182/blood-2018-99-111458 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2734-2734

Author(s):

Jochen Greiner ◽

Vanessa Schneider ◽

Hubert Schrezenmeier ◽

Markus Wiesneth ◽

Lars Bullinger ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Immune Responses ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Significant Inhibition ◽

Leukemic Cells ◽

Advisory Committees ◽

Oncology Research

Abstract Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies. Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays. Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction. CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays. In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. Disclosures Schrezenmeier: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

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Co-Targeting MCL-1 and BCL-2 Is Highly Synergistic in BH3 Mimetic- and Venetoclax/Hypomethylating Agent-Resistant and TP53 Mutated AML

Blood ◽

10.1182/blood-2020-141417 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Wenjing Tao ◽

Vivian Ruvolo ◽

Xuan Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Research Funding ◽

Single Agent ◽

Acquired Resistance ◽

Combination Group ◽

Bh3 Mimetics ◽

Bh3 Mimetic

Venetoclax (VEN), a highly selective BCL-2 inhibitor with limited single-agent activity in AML, has shown encouraging efficacy in combination with hypomethylating agents (HMA). Nevertheless, patients relapse and have limited treatment options. Like BCL-2, MCL-1 plays critical roles in the survival of AML cells and AML stem/progenitor cells. MCL-1 is also a known resistance factor to VEN. Preclinical studies have demonstrated that combined inhibition of BCL-2 and MCL-1 is highly effective in VEN-resistant AML cells. Diverse mechanisms contribute to the resistance to VEN, and likely also to BH3 mimetics targeting MCL-1 that are currently under clinical development in AML. The effectiveness of co-targeting BCL-2 and MCL-1 in the setting of various resistance mechanisms has not been fully explored. We investigated combined inhibition of BCL-2 and MCL-1 in AML cells resistant to apoptotic stimuli through various mechanisms and demonstrate that co-inhibition of BCL-2 with VEN and MCL-1 with AMG176 synergistically targets AML cells that exhibit intrinsic or acquired resistance to BH3 mimetics in vitro and in vivo. We generated AML cells with acquired resistance to VEN (VEN-R) or AMG176 (AMG-R) by exposing the cells to increased doses of the drug and we also generated the cells genetically overexpressing BCL-2, MCL-1, or BCL-2A1. We found that both VEN-R and AMG-R MV4-11 cells expressed increased levels of MCL-1, BCL-2, and BCL2A1, but decreased BAX. Although BCL-XL levels decreased in AMG-R MV4-11 cells, BAK, PUMA, and BID levels were also markedly lower in these resistant cells compared to the parental controls. VEN or AMG176 as single agents had diminished activity against AML cells with acquired resistance not only to VEN, but also to AMG176 and AML cells genetically overexpressing MCL-1, BCL-2, or BCL2-A1. In addition, we found that TP53 mutated primary AML cells expressed low levels of BAX and that Molm13 cells acquired a TP53 mutation (R248W) expressed lower levels of BAX and were more resistant to VEN, consistent with clinical observations, and they were also more resistant to AMG176. However, when VEN and AMG176 were combined, synergy was observed (combination index < 1). We next treated AML patient samples and found that combined inhibition of BCL-2 and MCL-1 was synergistic in primary AML cells and stem/progenitor cells obtained from patients with various cytogenetics/mutations, including TP53 mutations, and from patients resistant to/relapsed from VEN- or VEN/HMA-based therapy, even when AML cells were co-cultured with bone marrow-derived mesenchymal stromal cells that mimic the bone marrow microenvironment. To demonstrate potential clinical relevance, we developed a PDX model from a clinically-acquired VEN/HMA resistant AML patient and treated the PDX-bearing mice with VEN, AMG176, and the combination. Remarkably, the combination of VEN and AMG176 demonstrated strong antileukemia activities, markedly diminished not only AML blasts but also AML stem/progenitor cells, as determined by CyTOF analysis, and significantly extended survival (median 336 vs 126 d for controls, P < 0001), while VEN (129 d) alone and even AMG176 (131 d) alone had minimal efficacy. Several mice in the combination group survived over 400 d and died probably from old age with only minimal residual leukemia. In conclusion, we demonstrate the alteration of multiple BCL-2 family proteins contributes to BH3 mimetic resistance that can be overcome by combined inhibition of MCL-1 and BCL-2. The striking effectiveness of co-targeting BCL-2 and MCL-1 in AML resistance to a BH3 mimetic via various mechanisms or to VEN/HMA suggests broad clinical applications of this strategy, and warrants clinical evaluations. Disclosures Carter: Amgen: Research Funding; Ascentage: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding.

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Respective Place of Venetoclax and MCL1 BH3 Mimetics in Multiple Myeloma Treatment

Blood ◽

10.1182/blood-2018-99-109987 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 953-953

Author(s):

Patricia Gomez-Bougie ◽

Sophie Maïga ◽

Benoit Tessoulin ◽

Jessie Bourcier ◽

Antoine Bonnet ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Board Of Directors ◽

Ex Vivo ◽

Myeloma Cell ◽

Advisory Committees ◽

Bh3 Mimetics ◽

Promising Therapeutic Approach ◽

Apoptotic Proteins ◽

Bh3 Mimetic

Abstract Targeting anti-apoptotic proteins of the BCL2 family by BH3 mimetics is a new promising therapeutic approach in multiple myeloma (MM). The specific BH3 mimetic targeting BCL2, BCLXL or MCL1 trigger apoptosis and exploit the dependency on these different anti-apoptotic proteins to kill tumor cells. Because MM is mostly considered as dependent on MCL1, the recent clinical availability of MCL1 BH3 mimetics underlines an urgent need to better define patients that would benefit from a MCL1 targeted therapy. In the present study, we used a BH3 mimetic toolkit that includes venetoclax, A1155463 and A1210477, which target BCL2, BCLXL and MCL1 respectively to define dependencies/co-dependencies in a large cohort of 60 myeloma patients (21 at diagnosis and 39 at relapse). Alternatively, MCL1 dependency was confirmed using the S63845 MCL1 inhibitor in MM patient samples. Mononuclear bone marrow/blood cells were treated overnight with the respective BH3 mimetic and cell death was specifically measured in the tumor cell population. Primary MM cells dependencies were stratified using PCA analysis in three groups as highly dependent, intermediately dependent or not dependent. Our study demonstrated that half of patients at diagnosis were BCL2 dependent while only 10% were BCLXL dependent. The dependence on BCL2 or BCLXL was not significantly different between samples at diagnosis and relapse. Strikingly, we found that the MCL1 dependency was 33% at diagnosis while it was 69% at relapse, suggesting a significant increase in MCL1 dependency during the disease progression (p=0.01). Besides, 36% of overall patients showed co-dependencies on BCL2/MCL1. We also identified primary MM cells that did not depend on any of the three pro-survival molecules, both at diagnosis and relapse. Among this cohort of MM patients, 47 samples were further analyzed for the presence of recurrent translocations (t(11;14), t(6;14), t(4;14) and t(14;16)) allowing the analysis of dependencies in the different subgroups; these recurrent translocations lead to the overexpression of CCND1, CCND3, MMSET and MAF oncogenes, respectively. We found that BCL2 dependency was significantly higher in CCND1 subgroup (83%) compared to all other subgroups (20%, p=0.008). We also confirmed the BCL2/BCLXL mRNA ratio as a valuable biomarker to define BCL2 dependence (p=0.0001). At diagnosis, MCL1 dependency was absent in patients not harboring the common recurrent translocations while at relapse 6 out 9 patients not harboring the recurrent translocations were MCL1 dependent, indicating an increase of MCL1 dependency at relapse in this subgroup (p=0.03). Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection of cell death observed by BAK knock-down and the complete disruption of MCL1/BAK complexes upon A1210477 treatment, observed in MM cell lines and also in a patient sample. Interestingly, this complex was also dissociated in A1210477 resistant cells but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. Thus, BCLXL may act as a sink to bind freed pro-apoptotic proteins from MCL1 and limits MM cell death triggered by the specific targeting of MCL1. In conclusion, our study highlights the potential clinical use of BH3 mimetics in MM treatment guided by the practical ex-vivo testing of myeloma cell dependencies using the BH3 toolkit. This strategy could be used to identify the respective and tailored use of venetoclax, MCL1 BH3 mimetics or their combination in myeloma treatment. Disclosures Moreau: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML

Blood ◽

10.1182/blood.v128.22.101.101 ◽

2016 ◽

Vol 128 (22) ◽

pp. 101-101 ◽

Cited By ~ 3

Author(s):

Qi Zhang ◽

Lina Han ◽

Ce Shi ◽

Rongqing Pan ◽

Man Chun John MA ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Research Funding ◽

Acquired Resistance ◽

Resistant Cell ◽

Mitochondrial Activity ◽

Mitochondrial Oxidative Phosphorylation ◽

Expression Of Genes ◽

Apoptotic Proteins ◽

Resistant Cells

Abstract ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models. First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells. To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G). GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function. In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance. Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

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Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Blood ◽

10.1182/blood-2021-150191 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3-3

Author(s):

Alba Rodriguez-Meira ◽

Haseeb Rahman ◽

Ruggiero Norfo ◽

Wei Wen ◽

Agathe Chédeville ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Wild Type ◽

Advisory Committees ◽

Tp53 Mutations ◽

Molecular Landscape ◽

Mutant Cells

Abstract In myeloid malignancies, presence of 'multi-hit' TP53 mutations is associated with lack of response to conventional therapy and dismal outcomes, particularly when found in combination with a complex karyotype. Therefore, it is crucial to understand the biological basis of TP53-mutant driven clonal evolution, suppression of antecedent clones and eventual disease transformation to inform the development of more effective therapies. Myeloproliferative neoplasms (MPN) represent an ideal tractable disease model to study this process, as progression to secondary acute myeloid leukemia (sAML) frequently occurs through the acquisition of TP53 missense mutations. To characterize tumor phylogenies, cellular hierarchies and molecular features of TP53-driven transformation, we performed single-cell multi-omic TARGET-seq analysis (PMID: 33377019 & 30765193) of 22116 hematopoietic stem and progenitor cells (HSPCs) from 35 donors and 40 timepoints (controls, MPN in chronic phase, pre-AML and TP53-mutated sAML; Figure1a). TARGET-seq uniquely enables single-cell mutation analysis with allelic resolution with parallel transcriptomic and cell-surface proteomic readouts. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type alleles upon transformation, including parallel evolution of separate TP53 "multi-hit" subclones in the same patient (n=4/14) and JAK2-negative progression (n=2/14). Complex clonal evolution driven by chromosomal abnormalities (CAs) was present in all patients and TP53 multi-hit HSPCs without CAs were rarely observed. Subclones with recurrent CA such as monosomy 7 showed upregulation of RAS-associated transcription and preferentially expanded in xenograft models. Together, these data indicate that TP53 missense mutation, loss of TP53 wild-type allele and cytogenetic evolution are collectively required for leukemic stem cell (LSC) expansion. Integrated transcriptomic analysis of sAML samples (Figure1b) revealed three major populations: (1) a TP53-mutant cluster (Figure1c) characterized by an erythroid signature (e.g. KLF1, GATA1, GYPA; an unexpected finding as no cases showed diagnostic features of erythroid leukemia), (2) an LSC TP53-mutant cluster (Figure1d) and (3) a TP53-WT preleukemic cluster (Figure1e). The LSC cluster showed dysregulation of key stem cell regulators, from which we derived a novel 48-gene LSC score with prognostic impact in an independent AML cohort (HR=3.13; Figure1f). Importantly, this score was predictive of outcome irrespective of TP53 status for both de novo and sAML, demonstrating its broader potential clinical utility. TARGET-seq analysis uniquely allowed us to characterize rare TP53-WT preleukemic cells (preLSCs), which were almost exclusively confined to the immunophenotypic lineage-CD34+CD38-CD90+CD45RA- HSC compartment. PreLSC from sAML samples presented increased stemness, increased quiescence, aberrant inflammatory signaling and differentiation defects (Figure1g) as compared to HSCs from normal or MPN donors, both at the transcriptional and functional levels through in vitro long-term and short-term cultures. This indicates cell-extrinsic suppression of residual TP53-WT hematopoiesis. Longitudinal analysis of TP53-heterozygous mutant HSPCs at different stages of disease evolution (Figure1a) revealed that aberrant inflammatory signalling (e.g. BST2, IFITM1, IFITM3) in the genetic ancestors of TP53 "multi-hit" LSCs, but not the presence of TP53-mutations alone, was predictive of subsequent transformation. In a mouse model system, TP53-mutant cells challenged with sustained inflammatory stimuli acquired a mean 3-fold competitive advantage in WT: TP53 R172H/+chimeras. This indicates that pro-inflammatory cues from the tumour microenvironment promote fitness advantage of TP53-mutant cells whilst supressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis of the genetic, cellular and molecular landscape of TP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia (Figure1h). Since TP53 is the most commonly mutated gene in human cancer, we anticipate these findings will be of broader relevance to many other cancer types. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment. Drummond: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mead: Abbvie: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau.

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Inhibition of Mcl-1 Enhances the Efficacy of Tyrosine Kinase Inhibition in FLT3 Mutated AML and Synergizes with Venetoclax Targeting AML Stem Cells

Blood ◽

10.1182/blood-2019-125725 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1267-1267 ◽

Cited By ~ 1

Author(s):

Bing Z Carter ◽

Wenjing Tao ◽

Po Yee Mak ◽

Qi Zhang ◽

Xiangmeng Wang ◽

...

Keyword(s):

Cell Death ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Board Of Directors ◽

Median Survival ◽

Research Funding ◽

Cancer Center ◽

Advisory Committees ◽

Equity Ownership

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed limited clinical efficacy in AML as a single agent. FLT3 is the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients can adapt to TKI treatment by reactivating the MEK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 can be induced by VEN treatment and could be a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML and synergize with VEN, targeting AML cells and stem/progenitor cells. We treated FLT3-ITD positive AML cells with a selective inhibitor of Mcl-1 (AMG 176) and FLT3 TKIs and found that inhibition of Mcl-1 induced cell death and significantly enhanced the activity of sorafenib or gilteritinib in cell lines including cells acquired resistance to VEN (CI<1). It also enhanced the activity of sorafenib against blasts and stem/progenitor cells from primary AML samples harboring FLT3-ITD mutations. We previously showed that overexpression/knockdown of Mcl-1 greatly protected/sensitized AML cells from VEN induced cell death (Carter BZ, ASH 2018) supporting Mcl-1 as a key VEN resistance factor. We treated primary AML cells (n=5) with VEN (10 nM) or AMG 176 (250 nM) alone, or in combination and found that VEN+AMG 176 synergistically induced cell death in AML blasts and AML stem/progenitor cells even in samples clinically resistant to or relapsed after VEN containing regimen (CI<1). This synergism was also observed under mesenchymal stromal cells co-culturing conditions, while the combination was less toxic to normal bone marrow (NBM) cells (n=3) at even higher concentrations (VEN 20 nM, AMG 176 500 nM): apoptosis rate was at 82.4% or 80.8% under MSC co-cultures with AML blasts vs 34.2% or 36.4% under co-culture with NBM CD34+ cells. To investigate the antileukemia activity in vivo, we tested combined inhibition of Mcl-1 and Bcl-2 using two PDX models in NSG mice. The first model was developed from a resistant/relapsed patient with FLT3-ITD mutation and complex karyotype. The combination showed the most significant antileukemic activity and extension of survival, followed by AMG 176 and VEN treatment alone (median survival for the combination, 146 d, p=0.004; AMG 176, 137 d, p=0.032; VEN, 102 d, p>0.05 vs. control, 85.5 d; respectively). The second PDX model was developed from a patient who first responded and then became resistant to the combination of VEN and decitabine and harbors FLT3-ITD, NRAS, and GATA2 mutations and complex karyotype. VEN or AMG 176 monotherapies marginally prolonged survival (median survival 127 or 129 vs. control 124 d). The combination was highly effective in this model and greatly decreased circulating blasts (Fig. 1) and leukemia tissue infiltration, measured by flow cytometry and spleen size. CyTOF analysis demonstrated that only the combination strongly reduced blasts as well as the AML stem/progenitor cell populations. Median survival for the combination group currently has not been reached (>325 d) (Fig. 2). Collectively, these data demonstrate that inhibition of Mcl-1 enhances the efficacy of TKIs in FLT3 mutated AML. Furthermore, it synergizes with VEN, targeting not only AML blasts but also AML stem/progenitor cells, both in vitro and in vivo in PDX models with the potential of significantly improving treatment outcome, which warrants clinical evaluation. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Kisoji: Consultancy, Honoraria; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Ablynx: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding. Caenepeel:Amgen Inc.: Employment. Canon:Amgen Inc.: Employment. Hughes:Amgen Inc.: Employment. Morrow:Amgen Inc.: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

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Combination of Tyrosine Kinase Inhibitor with β-Catenin/CBP Modulator C82 Reverses TKI Resistance, Eradicates Quiescent CML Stem/Progenitors Cells, and Overcomes MSC-Associated Microenvironmental Protection

Blood ◽

10.1182/blood.v124.21.401.401 ◽

2014 ◽

Vol 124 (21) ◽

pp. 401-401 ◽

Cited By ~ 1

Author(s):

Hongsheng Zhou ◽

Po Yee Mak ◽

Hong Mu ◽

Duncan H Mak ◽

Hiroyuki Kouji ◽

...

Keyword(s):

Cell Death ◽

Tyrosine Kinase ◽

Western Blot ◽

Progenitor Cells ◽

Signaling Pathway ◽

Cell Lines ◽

Research Funding ◽

Critical Role ◽

Quiescent Cells ◽

Tki Resistance

Abstract Bcr-Abl tyrosine kinase inhibitors (TKIs) are effective in inducing remissions and improving survival in patients with CML but do not eliminate CML leukemia stem cells (LSCs). Wnt/β-catenin pathway is established to be active in CML and essential for CML LSC, while adult HSCs do not require fully active β-catenin for maintenance. Furthermore, Wnt/β-catenin signaling pathway plays a critical role in TKI resistance and stromal-mediated microenvironmental protection for CML stem and progenitor cells. We propose that combinations of β-catenin inhibitors and TKIs represent a potentially effective therapy by targeting both CML LSCs and leukemia-mediated microenvironmental protection. C82 is a novel β-catenin/CBP modulator that via binding to CBP inhibits the interaction of β-catenin and CBP and thus disrupts Wnt/β-catenin/CBP mediated cell proliferation and self-renewal signaling. CML cell lines and primary CML-BC patient samples were treated with combinations of C82 with different TKIs, including imatinib (IM), nilotinib (NIL), dasatinib (DAS), and ponatinib (PON). Both TKI-sensitive KBM5 (IC50=0.50±0.06µM, EC50=0.32±0.01µM, 48h) and TKI-resistant KBM5-STIT315I (IC50=1.44±0.06µM, EC50=0.36±0.09µM, 48h) cells were sensitive to C82. C82-TKI combinations synergistically induced apoptosis (C82-NIL, CI=0.30±0.07 and C82-DAS, CI=0.20±0.01 in KBM5; C82-NIL, CI=0.24±0.09 and C82-DAS, CI=0.36±0.05 in KBM5-STIT315I at 48h; respectively) and inhibited cell grwoth in both cell lines. KBM5, KBM5-STIT315I and K562 were co-cultured with normal human bone marrow derived-MSCs. Western blot showed that CML/hMSCs co-cultures increased β-catenin, CD44, and survivin proteins in CML cell lines. C82-TKI combinations induced similar degrees of cell death and proliferation inhibition with or without hMSC co-cultures, indicating the combination strategy can overcome MSC-mediated microenvironmental chemoprotection in CML. Western blot analysis showed that C82 significantly inhibited CD44 and survivin expression which was further reduced by C82-TKI combinations in KBM5 and KBM5-STIT315I cells. C82-TKI combinations were evaluated in CML sample (n=6) from heavily-treated and TKI-resistant CML-BC patients. Four out of 6 sample harbored BCR-ABL kinase mutations, including T315I, E255K/V, and H396R. Mononuclear cells from the patients were stained with cell division tracking dye CFSE and then co-cultured with hMSCs. Flow cytometry was performed to identify CD34+CFSEbright and CD34+CFSEdim cells, as quiescent and proliferating population, respectively. When CML cells were treated without hMSC co-culture, C82-TKI combinations exerted stronger synergistic effects in CFSEbright quiescent cells (CI=0.21±0.06, 0.29±0.07, 0.48±0.15, or 0.26±0.03 for combination of C-82 with IM, NIL, DAS, or PON) compared with CFSEdim proliferating cells (CI=0.43±0.05, 0.43±0.17, 0.50±0.20, or 0.44±0.06 for combination of C-82 with IM, NIL, DAS, or PON; respectively). While under co-culture conditions, similar levels of synergy was observed in proliferating (CI=0.39±0.02, 0.23±0.02, 0.32±0.05, or 0.27±0.01 for combination of C-82 with IM, NIL, DAS, or PON) and quiescent cells (CI=0.23±0.02, 0.20±0.01, 0.39±0.10, or 0.20±0.04 for combination of C-82 with IM, NIL, DAS, or PON; respectively). C82-TKI combinations also synergistically induced cell death in CD34+38- CML cells (n=4) and yielded minimum effect on normal bone marrows CD34+ cells (n=3). Invivo studies are ongoing with immunodeficient NOD/SCID/IL2rγnull mice injected with CML-BC patient samples. An open-label, dose-escalation phase I/II study of PRI-724 (active metabolite of C82) for advanced myeloid malignancies (NCT01606579), including CML patients in combination with dasatinb, is enrolling patients at MD Anderson Cancer Center and other centers. Our data demonstrate that β-catenin/CBP signaling pathway plays a critical role in quiescent CML stem/progenitor cells and disruption of the β-catenin/CBP interaction with C82 could overcome MSC-mediated microenvironmental protection for not only proliferating but also quiescent stem/progenitor cells in CML. Combinations of β-catenin/CBP signaling pathway modulator C82 with TKIs represent a potentially promising strategy to tackle TKI resistance and eradicate CML stem/progenitors cells and should be further investigated in larger studies. Disclosures Kouji: PRISM Pharma Co., Ltd: Employment. Cortes:PRISM Pharma Co., Ltd: Clinicaltrial PI for NCT01606579 Other. Andreeff:PRISM Pharma Co., Ltd: Research Funding. Carter:PRISM Pharma Co., Ltd: Research Funding.

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Co-Targeting Intrinsic and Extrinsic Apoptosis to Maximize Cell Death Induction in Venetoclax-Resistant AML Cells

Blood ◽

10.1182/blood-2021-148127 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3332-3332

Author(s):

Bing Z Carter ◽

Wenjing Tao ◽

Lauren B Ostermann ◽

Hong Mu-Mosley ◽

Po Yee Mak ◽

...

Keyword(s):

Cell Death ◽

Research Funding ◽

Single Agent ◽

Acquired Resistance ◽

Triple Combination ◽

Publicly Traded ◽

Family Protein ◽

Extrinsic Apoptosis ◽

And Control ◽

Mutant Cells

Abstract Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations. We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments). We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p<0.01) was more effective, and APG2575 plus APG115 (180 d, p<0.01) most effectively extended survival compared to each drug alone. Triple combination treated mice lived longest (185 d), which was significantly longer than APG115 plus APG1387 and APG2575 plus APG1387 but did not reach statistical significance compared to APG2575 plus APG115. Data showed that triple and APG115+APG2575 combinations were most effective, followed by APG2575+APG1387, then APG115+APG1387, APG2575, or APG115, and finally APG1387. Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased. In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

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DNA nick end labeling: a reliable marker for apoptosis?

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141184 ◽

1995 ◽

Vol 53 ◽

pp. 962-963

Author(s):

Anne F. Bushnell ◽

Sarah Webster ◽

Lynn S. Perlmutter

Keyword(s):

Cell Death ◽

Cultured Cells ◽

Apoptotic Cell ◽

Morphological Characteristics ◽

Detection Methods ◽

Tissue Preparation ◽

Preparation Methods ◽

Dna Laddering ◽

Disease States ◽

Reliable Marker

Apoptosis, or programmed cell death, is an important mechanism in development and in diverse disease states. The morphological characteristics of apoptosis were first identified using the electron microscope. Since then, DNA laddering on agarose gels was found to correlate well with apoptotic cell death in cultured cells of dissimilar origins. Recently numerous DNA nick end labeling methods have been developed in an attempt to visualize, at the light microscopic level, the apoptotic cells responsible for DNA laddering.The present studies were designed to compare various tissue processing techniques and staining methods to assess the occurrence of apoptosis in post mortem tissue from Alzheimer's diseased (AD) and control human brains by DNA nick end labeling methods. Three tissue preparation methods and two commercial DNA nick end labeling kits were evaluated: the Apoptag kit from Oncor and the Biotin-21 dUTP 3' end labeling kit from Clontech. The detection methods of the two kits differed in that the Oncor kit used digoxigenin dUTP and anti-digoxigenin-peroxidase and the Clontech used biotinylated dUTP and avidinperoxidase. Both used 3-3' diaminobenzidine (DAB) for final color development.

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